Method of propagation of culture yeast



Patented Mar-14, 19 39 i I Q 1] UNITED. STATES ATEN I I 2.156.329 i I IEdmund Leith Kitameyer, 'BloomfleluLN. J.

No Drawing. Application November 3', 1934, 7 serial No. 751,281

1 Claim. "(01. 195-82) "This invention relates to a method ofcontransferred to a fresh medium or may be kept trolling the growth andnutrition .of microfor some time in a cool place before transfer toorganisms used in' industrial processes. It reanother similar tube, orto the vessel in which is lates more particularly to the growth,nutritibnal started the first stageof the yeast manufacturing andenvironmental influences upon yeasts, parprocess. As the yeastgrows onthe surface of the 5 ticularly culture yeasts used to initiate themanusolid medium, the increasing cells pile on top of facture of acommercial compressed yeast, such oneanother and crowd along at thesides of the as is used in baking. colony so that the individual 'cellsof the culture An object of this invention is to maintain the aresubjected to a changing and wide variety of 10 mostdesirablephysiological state of the cultured conditions of. moisture,nutrient 'concentration, 10

microorganism, with asimple manner of manipualcohol, waste products, airand carbon dioxide. lation which does not greatly risk contaminationMost of the cells are subjected during most of of pure cultures byextraneous organisms. their existence as units to an environment ofAnother object of this invention is to bestow small amounts of nutrientand large amounts of upon culture'yeast such nutritional and physioyeastmeltabolic waste products and are not in 15 logical properties that-whenit is subsequently a state of active reproduction.. increased inquantity by any of the common -If liquid media are employed, the liquidconmanufacturing processes, the resultant large taini'ng all essentialyeast nutrients at a concenquantity of yeast will'be-of a better yieldand an tration of from 8 Balling to Bailing is main- 20 improvedquality. tained in' sterile condition" in tubes or other 20 Anotherobiectof this invention is to accomvessels. An inoculation of yeastculture is added plish these aims without any extra cost or effort witha platinum wire or sterile pipette and the more than that which isexpended in previously tube is kept at a proper temperature until theknown methods of handling cultures of indusgrowth of yeast is complete.If it is desired to trial Organisms. keep this culture several daysbefore the next 25 Still another object of this invention is totransfer, the culture is sometimes placed in a treat yeast in such amanner that its hereditary refrigerator. Under these conditions theyeast tendencies will be so altered that it may be incells at firstreceive an excess of nutrient but as creased in quantity in excess ofseveral. billion growth continues they are subjected for long times andstill maintain improved characteristics. periods to an environment inwhich there is little so At one time in the yeast manufacturing in--nutrient, large amounts of alcohol and other ry itwas customary toinitiate the growth of yeast metabolic waste products, as well as the y'by p n a small portion of previously anaerobic condition caused bysuper-saturation manufactured yeast .in a suitable quantity, of I of theliquid with carbon dioxide.

36 nutrient n as t e ye t in q t y With the older methods of caring forbaker's the regular manufacturing P ocess. Due to the yeast culturesthere are apt to appear, amongst fact that this often caused the yeastto become the normal cells, an occasional large, round, very infectedwith Wild yeasts and er Or anisms. granular cell which has a diameter offrom two this method has been generally abandoned. to five'times that ofthe normal cells, and some- 4" The present practice of handling yeast tobe times there appear some abnormal, elongated, 40

used to start the manufacturing process is similar very granular cells.These dissociated rough to that employed in the routine handling ofother cells, when obtained in pure culture produce a bacteriologicalcultures. The pure culture of gray appearing yeast of. slow growth rate,fOlIliyeast is obtained by known methods, and is kept ing unusually finebubblesoffoamin usual media.

4.3 alive by transfer-in liquid media such as malt, These cellshaveatendency to autolyze, and when 5 other grain, or molassessolutions, or by growth used to start a yeast manufacturing processproon solid media preparations of agar or gelatine. duce a compressedyeast 'of. poorer yield and The media are sterilized and the techniqueof slower dough fermentation time,-though -sometransfer is thatwell'known to bacteriologists. times producing bread of finer grain.This yeast If solid media are employedthey are kept, in is of somewhatdarker color and has an in- 5 a tube or small vessel protected frominfection creased tendency toward grittiness when comby a cotton plug,The yeast is introdu'oedQn very pressed. 1 have found that even if thecells resmall quantity on a' 'platinnm wire, and the main of thesmooth,apparentlyinormal-type they medium is kept at proper temperatureuntil yeast are subject to the influence of their environment growthis'complete. Then .tlie: -'culture maybe and retain certainpeculiarities derived there-. 55

from, for countless generations of reproduction under new conditions.

I have discovered a method of caring for yeast cultures which willmaintain the yeast in such a condition that when the culture is-used tostart a manufacturing process the resultant com-.- pressed yeast willshow a higher yield, a faster dough fermentation time, a better keepingqual-- ity and a better color. I have further found a simple method ofmaintaining baker's yeast cultures under such conditions that they willmaintain indefinitely their ability to reproduce into compressed yeastof the desired qualities.

In the practice of my invention we may use the regular liquid mediatubes now employed in the growth of cultures, for example a 13 Ballingmalt wort .tube adjusted to a pH of 5.00. Apure strain culturemay beused or a mixed culture such as fRasse M" may be employed. A platinumwire inoculation is placed in the tube and incubated at any of the usualtemperatures, for example 28 C. As soon as the first active evolution ofcarbon dioxide appears, the tube is agitated to drive off excess carbondioxide and ta straight platinum wire inoculation is made into a freshtube; and this in turn is transferred as soon as carbon dioxide isactively produced.

This procedure may readily be adjusted so that into the tube beinginoculated. Or the time between transfers may be still further varied bychanging the incubation temperature or the quantity of the culturemedium. In no case have I found it desirable to cause the yeast to growuntil the nutrient is exhausted as is the common practice. By my partialgrowth method of transfer, the yeast is continuously held in a the cyclebetween transfers has elapsed before enough carbon dioxide has beenevolved to approach anaerobic conditions. Although the amount ofinoculating material is small, the lag period of growth rate is greatlyreduced due to the high state of activity of the inoculated cells.

The beneficial effects of this method of care of culture yeasts cannotbe explained entirely by the nutritional state of the culture, but mustbe attributed ratherto an evolutionary alteration, since the effects aredistinguishable somanygenerations after any chemical constituents wouldhave been diluted to the vanishing point by cell reproductions.

Thus if we take from one pure culture of yeast, two inoculations to berun in two sets of tubes containing the same media we may trace certainfundamental difiererices in efl'ects.- The one culture may betransferred by the old method, that.

is, transferred with a loop or pipette three or four times a week, theyeast being allowed to thoroughly grow out between each transfer, andthe other culture transferred byflmy partial growth method which I" havedescribed. As we carry these cultures we will note at time of transferthat the old method culture has cells tending to be rounded, vacuoledwith somewhat granular cytopiasms, and a number of dead cells may beseen as well as an occasional rough dissociated cell, while the partialgrowth culture noted at time of transfer has cells tending to elongatein active reproduction, showing smooth cytoplas'ms, no vacuoles and deadcells, and dissociated cells are almost entirely absent.

If we grow these cultures for four or five months, we may then use themto start a commercial yeast manufacturing process in which to compareresults. In the commercial process these two cultures will presentsimilar appearances except that the partial growth culture will tend toproduce a thicker, whiter foam containing more yeast. But in theresultant compressed yeast certain fundamental differences may be noted.If control of all steps is accurately maintained, the partial growthculture will be found to have produced from 1% to 4% more yeast than theold method culture. Chemical tests will show that the partial growthculture has been able to extract a greater percentage of nitrogen fromthe nutrient wort. The compressed yeast from the partial growth culturewill show a slightly improved color and slightly better keeping quality.Equal weights of the compressed yeast from these two cultures, when usedin representative types of dough, will show that the partial growthculture yeast will definitely shorten the dough fermentation time by aconsiderable percentage, and will produce a baked product of betterquality than will the compressed yeast from the old method culture.

This description of my partial growth method' sequently used, in anenvironment rich in sugar,

nitrogen, phosphate and all other yeast nutrients. It will be noted thatI present further novelty in the continuous maintenance of culture yeastin an environment very low in alcohol,

carbon dioxide, and other yeast waste products.

It will be seen that I present further novelty in keeping culture .yeastin a state of perpetual reproduction. It may be seen that I reveal a newdiscovery in the fact that yeast may be influenced intohereditary'changes of an evolutionary nature, and that the successfulapplication of these hereditary changes to a manufacturing be containedin the transfer made by any device which avoids infection. J o

Procedines in culture care, such as replacement of nutrient as it isused, physical or chemistraight platinum wire. The nutrient wortmay ysort of a flask or vessel and v cal removal, dilution or neutralizationof metabolic .waste products, or retardation of growth rate by lowvtemperatures, high .acid concentrations or unbalanced nutrients will beseen to comprise more cumbersome methods of employing some of theprinciples which I have explained.

.Partial growth cultures in flasks may be aerated by bubbling filteredair through the nutrient wort. If this procedure is followed, it is notpossible by simple observation to tell when the first active evolutionof carbon dioxide occurs, so that the time for transfer may bedetermined, but the following method may be followed with good results.A flask is inoculated with the culture, standard conditions of quantityof inoculating material, quantity and concentration of nutrient,temperature, and aeration being selected. The yeast is allowed topropagate'and the degrees Bailing of the nutrient wort are tested andrecorded from time to time. This test of the fermentation of the sugarwill supply mum-- ciently accurate standard upon which to base a regularprocedure of transfer. It will be noted that the destruction of nutrientandrlse in waste.

products of the medium occurs at a greatly increasing rate duringgrowth, due to the geometric progression of the number of yeast cells,and that yolatile waste products such as carbon di-, oxide and alcoholwill be partly removed by the passage of air through the flask. Due tothese facts, it is safe to transfer the culture, in accordance with thepartial growth principles, on a regular schedule atany time between thetime when the first definite drop in degrees Bailing occurs, and thetime when three quarters of the time from the setting to the time of thelowest degree Balling has elapsed. Thus if the nutrient in thestandardflask showed its first definite drop in Bailing at 10 hours and reachedits lowest degree Bailing at 32 hours, it would be proper to make theregular routine transfer of such flasks at any time between 10 and '21hours with-.

out any necessity of performing tests.

These principles may be applied to the continuance of culture yeast intothe early part of the plant manufacturing process, that is, the stepsused in the manufacture of seed yeast. Just how far this may be carriedefllclentl'y will depend upon the particular process and conditions inthe particular plant. As an illustration we may take c. c. ed at13Balllng, and add it to one'gallon of 13 Bailing nutrient wort. whenpartial growth has occm'red,'wemayaddthisto100gallonsof of a partialgrowth culture startaerated Balling nutrient wort, and this in turn whenpartially grown may be run into 800 gallons of aerated 6 Bailingnutrient. Before the yeast exhausts this sugar, this solution may beadded to 2,500 gallons of aerated 4 Bailing wort. When proper partialgrowth has taken place in this fermenting liquid, it may be used to set5,000 gallons of' aerated 3 Bailing wort. Here the yeast may be allowedto complete its growth, with subsequent addition of nutrient, or at thisor at any stage, the partial growth yeast may be filtered or separatedto start the next fermentation stage, and the unused nutrient of thewort maybe used as part of the nutrient for r a commercial yeastpropagation. Thus the partial growth principle may,be efllcientlyapplied in yeast manufacture, in any stage from the culture tube to thestart of the commercial yeast propagation.

This partial growth principle may be applied ilar success wherever theorganisms are used in any comparable function in industrial processes.Wherever bacteria or other fungi are used in considerable quantity toperform some chemical inv to other micro-organisms than yeast withsimdustrial function, thls'partial growth method of transfer of cultureswill be found valuable. In a What I desire to secure by United StatesLet-.

ters Patent is:

The method of propagating pure yeast cultures which comprises growingsaid cultures in a normal yeast nutrient solution, and repeatedlytransferring said cultures from solution to solution at a period betweenthe time the sugar first shows readily appreciable depletion and thetime when three-quarters of the growth time has elapsed. v p

